Unidirectional Permeability Through Intestinal Tissue

This assay is used to determine the unidirectional permeability of a test compound through intestinal tissue segments (duodenum, ileum, jejunum and colon)

Required from Customer

  • A study design—the customer must specify the value of the assay variables listed in the Options section
  • Either a minimum of 2.0 mL of test compound at 10 mM in DMSO, or
  • 50 mg of powder
  • The molecular mass (exact mass) of test compound and its salt form
  • The solubility of the test compound in Krebs Ringer Bicarbonate (KRB)buffer
  • The stability of the test compound in KRB buffer at 37°C for the duration of the experiment
  • The MSDS or handling and storage information, e.g., light sensitive, store at -20°C, etc.


  • Suitability results (solubility, chemical stability NSB)
  • The flux or apparent permeability of the test compound
  • The permeability of the control compounds
  • Donor demographics for human tissue studies


  • The test compound at 100 µM in KRB with maximum DMSO concentration of less than 2%

Assay System

  • One or more intestinal segments mounted in Ussing chambers and thermostatically controlled at 37°C

Assay Conditions

  • Solubility:
  • Single incubation target concentration 50-100 µM pH 7.4 (6.5 if applicable)
  • 37°C, equilibrate for max of 3 hours
  • Filter 3 aliquots (N=3) solution, dilute
  • Analyze using matched matrix and non-validated LC-MS/MS
  • Chemical stability:
  • Mid range concentration, assay matrix=pH 7.4 KRB incubation temp, 37°C, time points: 0 and 180 minutes
  • Analysis via LC-MS/MS-peak area response ratio recovery between 85%
  • and 115% of initial
  • Non-specific binding:
  • Three chambers (N=3) are used
  • Dose with a solution containing 1% of the concentration of the test compound that is in dosing solution used in the permeability experiment
  • Sample receiver side at 0 and 180 minutes
  • Unidirectional (mucosal-to-serosal) Permeability:
  • N=4/treatment/donor
  • Sampling: Donor: 0, 30, 60, 90, 120, 150 minutes,
  • Receiver: 30, 60, 90, 120, 150 minutes
  • The transport buffer is KRB at pH 7.4 in the basolateral chamber—other buffers may be used in the donor chamber
  • Unless the optional full method validation is ordered the concentrations of test compound are determined using a generic LC-MS/MS method with a minimum 5 point calibration curve

Assay QC

  • The integrity of the tissue system is verified by determining the permeability of Lucifer Yellow, Antipyrine and Atenolol that are co-dosed with the test compound


  1. The typical concentration of the test compound in the dosing solution is 100 µM. Factors to consider when selecting a dosing concentration are both the solubility of the test compound in KRB buffer and cytotoxicity.
  2. For reproducible results the test compound should be stable under assay conditions. Its solubility must also be greater than 100 µM in KRB buffer containing 2% DMSO. If the stability or the solubility of the test compound is unavailable then Absorption Systems will conduct this assessment at an additional charge.
  3. If the solubility of the test compound is less than 100 µM, even with the addition of DMSO to the KRB buffer, then options include running the permeability assay with 1% Bovine Serum Albumin (BSA) in the receiver side buffer.
  4. Non-specific binding is determined by measuring the percent recovery of the test compound from the Ussing chamber. Recovery below 70% can bias results. Because of this the customer is consulted when the percent recovery of the test compound is less than 70%.
  5. An optional complete analytical method development includes:
    1. Optimization of MS: Initial conditions will be established by infusing a solution containing the test compound into an appropriate mass spectrometer. The goals in MS optimization are to maximize detector response for the test compound and determine the approximate lower limit of quantitation (LLOQ) of the test compound.
    2. Optimization of HPLC: HPLC parameters will be optimized using an appropriate selection of LC column(s) and mobile phase. The goal in HPLC optimization is to develop appropriate chromatographic run-times while maintaining adequate levels of sensitivity.
    3. Optimization of Sample Preparation: Various means of sample preparation, e.g., direct injection, dilution with appropriate solution, protein precipitation, solid phase extraction etc., will be evaluated for their efficiency in extracting the test compound from the biological matrix. The goal in extraction optimization is to achieve the required lower limit of quantitation (LLOQ) while alleviating matrix suppression effects.
  6. An optional validation is performed using calibration standards and quality control (QC) samples containing the test compound at known concentrations. In order for the analytical method to be considered acceptable the results from at least one method validation run must meet the following criteria:
    1. The standard curve must include a minimum of five standards with their concentration back calculated to ±15% (±20% at the LLOQ) of their theoretical concentration when the correlation coefficient of the regression model is ≥0.990.
    2. The calculated concentration of at least two-thirds of all QC samples and at least one-third of the QC replicates must fall with ±15% of their respective theoretical concentration.
    3. The intra-assay coefficient of variation (CV) of the replicate determinations of the low, medium and high QC samples must not exceed ±15%, and the average concentration for each set of QC replicates as calculated using the standard curve must be within ±15% of their theoretical concentration.
    4. The lowest standard is accepted as the lower limit of quantitation if the typical response at this concentration is at least 5 times greater than any interference in the blank matrix at the retention time of the analyte(s).
    5. The response ratio of any interfering peak(s) in the matrix blanks at the retention time of the analyte(s) must be < 20% of the response to the LLOQ standard.


  1. The customer must specify:
    • either the basic, standard or custom report format
    • the species from which the intestinal segments will be harvested
    • the number of donors to be usedthe intestinal segments
    • the number of chambers per segment
    • the pH of the buffer solution on the donor side within the range of 5.5 to 8
    • the number of replicates run in the assay within the limits of the amount of tissue available from each segment—varies by species
  2. The customer can request:
    • complete analytical method development and pre-study validation
    • a different concentration of the test compound in the dosing solutions—the analytical sensitivity will influence the range of acceptable concentrations—depends upon the compounds
    • different sampling time points
    • that transporter inhibitors or metabolic inhibitors be added along with the test compound
    • stability of test compound can be assessed in intestinal S9 fractions for additional cost
    • upon request, tissue accumulation of test compound can be determined up to 2 weeks post experimental treatment for an added cost